FIG. 5.

Reduced HIV replication in ATM knockdown cells. (a) Inhibition of endogenous ATM and ATR protein expression by shRNA-producing lentiviral vectors. Results are shown for Western blotting of cellular lysates with anti-ATM, anti-ATR, and anti-Chk2 antibodies in ATM knockdown (ATMi), ATR knockdown (ATRi), and double-knockdown (DKD) P4.2 cells as well as in P4.2 cells transduced with a control (C) lentiviral vector. (b) Each line was infected with HIV-1 at an MOI of 0.5. HIV-1 replication was assayed by p24 ELISA with the culture supernatants 6 days later. (c) Inhibition of endogenous MRE11 protein expression by shRNA-producing lentiviral vector. MRE11 knockdown (MRE11i) P4.2 cells were infected with HIV-1 at an MOI of 0.5. HIV-1 replication was assayed by p24 ELISA with the culture supernatants 7 days later. (d) HIV-1 Tat-mediated transcription in ATM knockdown HeLa cells. Tat-expressing plasmid (100 ng) and HIV-1-LTR-luciferase (HIV-1-LTR-Luc) reporter plasmid (100 ng) were cotransfected into control P4.2 cells (P4.2:C) or ATM knockdown P4.2 cells (P4.2:ATMi) (2 × 10⁴ cells). A luciferase assay was performed 24 h later. Results are from three independent transfections. (e) Tat-expressing plasmid (100 ng) was transfected into P4.2 cells in triplicate. β-Gal activity of LTR-LacZ-containing cellular lysates was measured at an optical density at 570 nm 24 h later.